ว.วิทย. มข. 44(2) 272-281 (2559) KKU Sci. J. 44(2) 272-281 (2016) ความเป็นพิษของกลูโคนาสเตอติอินและสารอนุพันธุ์ ต่อเซลล์ MCF-7 และ HepG2 Cytotoxicity of Gluconasturtiin and Its Derivative against MCF-7 and HepG2 Nattaya Konsue1* and Ahmad Faizal Abdull Razis2 1Program in Food Technology, School of Agro-Industry, Mae Fah Luang University, Chiang Rai 57100, Thailand 2Food Safety Research Centre (FOSREC), Faculty of Food Science and Technology, Laboratory of UPM-MAKNA Cancer Research, Institute of Bioscience, Universiti Putra Malaysia, 43400, UPM Serdang, Selangor, Malaysia *Corresponding author, E-mail: [email protected] บทคัดย่อ มีผลงานวิจัยเผยแพร่อย่างกว้างขวางเกี่ยวกับความสามารถในการต้านมะเร็งของสารออกฤทธิ์ทางชีวภาพ ประเภทไอโซไธโอไซยาเนทในพืชตระกูลกะหล่ํา ในงานวิจัยนี้ได้ทําการศึกษาความสามารถในการเป็นพิษต่อ เซลล์มะเร็งเปรียบเทียบระหว่าง gluconasturtiin สารตั้งต้นที่พบตามธรมชาติใน watercress และสาร phenethyl isothiocyanate (PEITC) ซึ่งเป็นสารอนุพันธุ์ในเซลล์มะเร็งเต้านม (MCF-7) และเซลล์มะเร็งตับ (HepG2) โดยวิธี MTT assay ช่วงความเข้มข้นที่ศึกษาได้แก่ 0-100 μM และผันแปรระยะเวลาในการบ่มสารเป็น 24 48 และ 72 ชั่วโมง โดยมี paclitaxel เป็นตัวอย่างควบคุมที่เป็นบวก ผลการทดลองพบว่า การมีชีวิตของเซลล์ MCF-7 ลดลงเมื่อเลี้ยงด้วยอาหารที่มีสาร PEITC โดยความเข้มข้นที่ทําให้การมีชีวิตของเซลล์ลดลงร้อยละ 50 หรือ IC50 มีค่าเป็น 10.22 3.61 และ 2.68 μM ในขณะที่ค่า IC50 ของ PEITC ใน HepG2 มีค่าเป็น 6.74 4.22 และ 4.19 μM ตามลําดับ นอกจากนี้พบว่า gluconasturtiin ไม่มีความเป็นพิษต่อเซลล์มะเร็งทั้งสองชนิดในช่วงความ เข้มข้นที่ทําการศึกษา และเพื่อศึกษากลไกความเป็นพิษของ PEITC จึงได้ทําการทดสอบความสามารถในการทําให้ เกิดอะพอพโทสิสในเซลล์ MCF-7 โดยวิธี fluorescence microscopy ด้วยเทคนิคการย้อมด้วย AO/PI ผลการ ทดลองเลี้ยงเซลล์ MCF-7 ในสภาวะที่มี PEITC ที่ความเข้มข้นเท่ากับ IC50 เป็นเวลา 48 ชั่วโมง พบว่าโครมาติน ของเซลล์เกิดการจับกันแน่นและนิวเคลียสเกิดการแตกหัก และพบว่ากลไกการเป็นพิษต่อเซลล์ของ PEITC เกี่ยวข้องกับกระบวนการอะพอพโทสิส แต่อย่างไรก็ตามควรมีการศึกษาความเป็นพิษต่อเซลล์ปกติต่อไป งานวิจัย วารสารวิทยาศาสตร์ มข2 เลมท่ ี่ 44 ปที ี่ . 273 ABSTRACT The putative anticancer abilities of isothiocyanates, bioactive compounds in brassica vegetables, have been widely published. In the current study, gluconasturtiin, found naturally in watercress, and its derivative, phenethyl isothiocyanates (PEITC), toxicity against human breast cancer cells MCF-7 and hepatoma cell line HepG2 were determined employing MTT assay. A range of concentrations between 0-100 μM was used to study dose dependent cytotoxicity at 24, 48 and 72 hrs incubation periods. Paclitaxel at the same concentrations was served as a positive control. The results showed that in MCF-7 cell line, PEITC led to a time-dependent decrease in cancer cells viability with IC50 of 10.22, 3.61 and 2.68 μM at 24, 48 and 72 hrs, respectively, whereas those of HepG2 were 6.74, 4.22 and 4.19 μM. Moreover, gluconasturtiin had no effect on both cell lines at the concentration range studied. Apoptogenic effects of PEITC on MCF-7 were studied using fluorescence microscopy (AO/PI double staining). When cells were exposed to PEITC for 48 hrs at IC50 dose chromatin condensation and nuclear fragmentation were noticed. Moreover, apoptosis is one of chemopreventive mechanisms of PEITC which, however, toxicity on normal cells should be further investigated. คําสําคัญ: พืชตระกูลกะหล่ํา วิธี MTT assay ความเป็นพิษต่อเซลล์ กลูโคซิโนเลท Keywords: Brassica, MTT assay, Cytotoxicity, Glucosinolates INTRODUCTION the vacuoles and hydrolysed by cytosolic Cruciferous vegetables, i.e. watercress, myrosinase, a thioglucoside glucohydrolase Brussels sprouts, broccoli, cabbage, kai choi, enzyme (EC 3.2.3.1) (Nugon-Baudon and kale, horseradish, radish and turnip are Rabot, 1994). Gut flora such as Escherichia coli categorised within the brassica family and Bacteroides vulgatus, have also been (Brassicaceae). Recent scientific research has reported to contribute to dietary glucosinolate focused on the chemopreventive effect of hydrolysis (Rabot et al., 1993; Shapiro et al., cruciferous vegetables due to their high 1998). Myrosinase breaks the beta- content of beneficial substances, gluco- thioglucosidic bond of the glucosinolate sinolates and their metabolites, iso- molecule to produce glucose, sulphate and thiocyanates (ITCs) (Verhoeven et al., 1997). unstable aglycone intermediates, which then When the tissue of cruciferous plants is undergo non-enzymatic intramolecular disrupted by mechanical food processing or (Lossen) rearrangement to yield iso- masticating, glucosinolates are released from thiocyanates as shown in Fig. 1 (Fahey and 274 KKU Science Journal Volume 44 Number 2 Research Talalay, 1999). It has been reported that ITCs exert their chemopreventive activity by Gluconasturtiin enhancing the elimination of carcinogenic substances from the body, inducing apoptosis pH = 6.5 Myrosinase/H2O (programmed cell death) of pre-cancerous cells and by arresting cell proliferation. There Phenethyl isothiocyanate (PEITC) is evidence that the intact glucosinolate from broccoli was not active in modulating Figure 1 Generation of the breakdown xenobiotic-metabolising enzymes, and product phenethyl isothiocyanate (PEITC) incubation with myrosinase to promote from myrosinase-dependent gluconasturtiin degradation of the glucosinolates to ITCs was hydrolysis. necessitated (Vang et al., 2001). Furthermore, some studies demonstrated that some MATERIALS AND METHODS glucosinolates also have adverse effects by 1. Materials inducing DNA damage, which may develop to Glucosaturtiin and thioglucosidase cancerous cells (Conaway et al., 1999; IARC, from Sinapsis alba seed were a gift from CRA 2004; Robbins et al., 2005). There are numbers Agricultural Research Council, Italy. Roswell of research showed cytotoxicity of ITCs in Park Memorial Institute (RPMI)-1640 medium, different cell lines. However, the effects are DMSO, fecal calf serum (FCS) Trypsin-EDTA poorly understood in glucosinolates including solution, penicillin/streptomycin, thiazolyl gluconasturtiin which is the precursor of a blue tetrazolium bromide, acridine orange and potent anticancer compound phenethyl propidium iodide were purchased from Sigma- isothiocyanate (PEITC). aldrich (Singapore). Therefore, the current study was 2. Human cell lines aimed to investigate the cytotoxicity property Human cancer cell lines namely of gluconasturtiin in comparison with breast (MCF-7) and liver (HepG2) were grown phenethyl isothiocyanate. MTT assay was in RPMI-1640 supplemented with 10% FCS carried out in human breast cancer cells MCF- and 1% penicillin/streptomycin. The media o 7 and hepatoma cell line HepG2. Paclitaxel was stored in refrigerator (2-8 C) and warmed o was served as a positive control. to 37 C before use. The cell lines were o maintained at 37 C in a 5% CO2 atmosphere with 95% humidity prior to utilization in further steps. The remaining cells were stored งานวิจัย วารสารวิทยาศาสตร์ มข2 เลมท่ ี่ 44 ปที ี่ . 275 in cryopreservation medium containing 90 % positive control was plated in each well. In FCS and 10 % DMSO. case of PEITC, 0.3 unit/mL of myrosinase in 3. In vitro assay for cytotoxic activity water was prepared prior to 5 μL was added The cytotoxic potential of the test directly into gluconasturtiin containing material in human cancer cell lines were medium. Plates were incubated at various determined employing the method modified incubation periods (24, 48 and 72 hrs) at 37 oC from van Meerloo and colleagues (van and 5% CO2 and 95% relative humidity in a Meerloo et al., 2011). CO2 incubator. The procedure was replicated 3.1. Preparation of cell suspension three times. for assay 3.3. MTT assay Human cancer cell lines were grown Prior to treatment, the media were in 25 mL conical flasks at 37oC in an discarded and 20 μL of 5 mg/mL MTT atmosphere of 5% CO2 and 95% relative prepared freshly in PBS was added. Incubation humidity in complete growth medium to for 3 hrs at 37 oC in the dark was required. At obtain enough number of cells as described the end, MTT was discarded and 100 μL above. 1 mL of Trypsin-EDTA was added into DMSO added to start the reaction, absorbance 80% confluency in flask in order to harvest at 570 nm, which indicated viability of cells, cells. The flask was then incubated at 37 oC was used. Finally, % cells viability and IC50 for 5-7 min and detached cells were were calculated. collected. Viable cell density at 1 x 105 cells 4. AO/PI staining were plated in each well. 100 μL of cell AO/PI double staining method was suspension in complete growth medium was performed according to Guan and colleagues added into each well. The plates were (Guan et al., 2007). Acridine orange (AO) and incubated at 37 oC for 24 hrs in an propidium iodide (PI), nuclear staining dyes, atmosphere of 5% CO2 and 95% relative were employed. AO is permeable to live and humidity in an incubator. After incubation, the dead cells while PI enters only dead cells. test material and positive control were added. MCF-7 was selected to study the 3.2. Treatment of cells apoptotic effect of sample. Firstly, MCF-7 was Serum free RPMI medium containing grown in 25 mL flask for 48 hrs prior to 10 mL gluconasturtiin or paclitaxel (positive control) of serum free medium containing IC50 of at a range of 0-100 μM was prepared. The 200 PEITC was added into cells before incubation μL of RPMI in the presence of test material or at 37 oC for 48 hrs. AO and PI stock solutions 276 KKU Science Journal Volume 44 Number 2 Research at concentration of 1 mg/mL PBS were manner. IC50 (concentration causing death of prepared